Double strand breaks (DSBs) in genomic DNA can be caused by exogenous DNA damaging agents (e.g. ionizing radiation), but are also produced as intermediates in normal recombination events, including V(D)J recombination. DSBs are lethal, thus must be repaired for cells to live; aberrant DSB repair is also linked to frequent tumor development. The end-joining pathway for repair of DSBs simply involves joining broken DNA ends back together, modifying the ends as necessary to permit successful ligation. Genetic analysis has identified many factors involved in this pathway, including Ku, DNA-PKcs, XRCC4, DNA Ligase IV, and MRE11; many more factors will likely be added to this list. Most, if not all of these factors are required both for normal resistance to ionizing radiation as well as the joining of V(D)J recombination intermediates, but their molecular roles in these processes are not well understood. The goal of this proposal is thus to clarify the molecular mechanism of end-joining DSB repair. I. Can the identified factors cooperate to perform different end-joining functions in vitro? Most of the factors already linked to this pathway will be purified and employed in sensitive cell-free assays to determine if and how these factors cooperate to perform different functions in repair of a DSB. II. Identification of other proteins required for end joining by affinity purification. The in vitro properties of Ku argue that it may act to nucleate assembly of end joining complexes. This will be the basis for affinity purification of other, possibly as yet unidentified factors important for this pathway. III. How are end-joining complexes assembled in cells? The role of different end-joining factors in vivo will be studied by inducing a specific DSB in cells, "freezing" the proteins at this DSB, and identifying which proteins are found there, and when, during the course of induction and resolution of the DSB. IV. Determination of different molecular roles for Ku by structure: function studies. The requirements for Ku's functions both in vitro and in vivo will be identified by analysis of Ku mutants.